The aim of this research was to study the effect of sulphur (S) fertilization on oil biosynthesis and its related variables at various stages of seed development, and to find possible explanations for increased oil content in the seeds of mustard (Brassica juncea L. Czern and Coss) due to S fertilization. Acetyl-CoA carboxylase activity and contents of oil, acetyl-CoA, soluble protein, total RNA, total sugar and sulphur were determined in the developing seeds of mustard grown in the field with sulphur (+S) and without sulphur (-S). The period between 10 to 30 days after flowering was observed as the active period of oil accumulation in the developing seeds of mustard. The accumulation of the oil was preceded by a marked rise in acetyl-CoA carboxylase activity and acetyl-CoA concentration, which declined rapidly when oil accumulation reached a plateau. Total sugar content decreased, while protein content increased during the active period of oil accumulation in the developing seeds (i.e. between 10-30 days after flowering). Sulphur fertilization significantly (P < 0.05) enhanced the oil accumulation in the developing seeds at all the growth stages. The increase in the oil content was 5-63% with S fertilization over the control treatment. Acetyl-CoA carboxylase activity and contents of acetyl-CoA, soluble protein, total RNA and sugar were significantly (P < 0.05) higher in the developing seeds of +S-treated plants compared to -S-treated plants. It is suggested that the increase in the oil content with sulphur fertilization may be associated with the increases in acetyl-CoA carboxylase activity through the enhancement of acetyl-CoA concentration. Further, the increased sugar content due to S fertilization provided enough carbon source and energy for oil biosynthesis.